A new method for assaying polymerization of tubulin has been developed. The method, which utilizes the colchicine binding properties of tubulin, directly quantitates tubulin incorporation into microtubules. Under appropriate conditions cAMP and cGMP inhibit the in vitro assembly of microtubules from crude supernatants. cAMP has no effect on the polymerization of purified tubulin. In an effort to determine the mechanism of the cAMP-dependent inhibition of polymerization, the effects of cAMP on protein phosphorylation in the crude supernatant and preparations of purified tubulin habe been studied. ATP, ADP, AMP, and adenosine have inhibitory effects on the polymerization of microtubules from crude supernatants of brain homogenates. The mechanisms of these effects are under study.